P1398: INSIGHT INTO ACUTE MYELOID LEUKEMIA INDUCED MSC SECRETOME TO REROUTE THE COURSE OF LEUKEMIA BONE MARROW NICHE
Authors: G. Borella, A. Da Ros, G. Borile, G. Longo, E. Campodoni, M. Montanari, M. Benetton, M. Sandri, C. Tregnago, F. Locatelli, M. Pigazzi
Journal: HemaSphere (2022)

Background: Bone marrow (BM) niche sustains hematopoiesis and hematopoietic stem cells (HSCs) homeostasis, but at Acute Myeloid Leukemia (AML) onset it undergoes to drastic remodeling to support the disease, thus impeding HSCs functions. To date, the underlying mechanisms and interactions within BM niche are not clear, but several line of evidences suggest that mesenchymal stromal cells (MSCs)-derived soluble factors play a key role in suppressing HSCs activity strongly contributing to the emergence of an AML-infiltrated BM niche thus increasing treatment complications and mortality rates. Aims: We aim to characterize the secretome profile of MSCs derived from BM of AML patients at diagnosis (AML-MSCs) with respect to MSCs derived from healthy donors (h-MSCs), and h-MSCs when co-cultured with AML blasts (induced, iAML-MSCs) in a predictive three-dimensional (3D) AML-niche model, including the mechanism whereby normal hematopoiesis is impaired and leukemia advantaged. Methods: Gene expression of AML-MSCs and iAML-MSCs showed an enrichment of upregulated genes related to inflammation and tumor-associated soluble factors with respect to h-MSCs. We explored AML-, h- and iAML-MSCs secretome in 3D by a SILAC-label proteomic analysis. Then, we dissected the role of 3 candidate secreted proteins by evaluating the effects when exogenously expressed in AML or HSCs co-cultures (CD34+ sorted cells), and explore their role during chemotherapy treatment. Results: 3D co-cultures of h- or AML-MSCs and AML blasts or HSCs showed that AML-MSCs strongly supported AML cells proliferation (by Ki67), but decreased normal HSCs proliferation (reduction of 32%). Moreover, we observed an increased number of CD34+ colony-forming cells (CFCs) when co-cultured with AML-MSCs with respect to h-MSCs, suggesting the maintenance of more immature hematopoietic cells. Same effects on CD34+ block of differentiation was observed when co-cultures of AML-MSCs and HSCs were separated via a transwell-insert. These latter findings suggested that AML-MSCs secretome exerted a suppressive role on HSCs regulation. We applied Mass-Spectometry analysis of AML blasts and AML- or h-MSCs co-cultured for 21 days in 3D, finding a total of 15 differentially new synthesized secreted factors in the 3D medium of co-cultures with the AML blasts, related to inflammation, angiogenesis and cancer pathways. Through a validation by ELISA assay, we selected PAI-1, CHI3L1 and PTX3 as the 3 top up-secreted factors in the AML niche for further functional studies. Recombinant proteins PAI-1, CHI3L1 and PTX3, used in combination or alone, were shown to reduce CD34+ proliferation (of 30% when used in combination, and 25%, 17% and 10%, respectively when used alone), including the support of a more immature phenotype (CD34+/CD33-) than when cultured alone or with h-MSCs. Interestingly, adding the recombinant proteins to the co-culture of CD34+ with h-MSCs we recovered same features mediated by AML-MSCs in terms of proliferation (reduced of 15%), CFCs (2-fold increase) and CD34+ expression (increased of 17%). However, recombinant proteins did not showed effects on AML blasts neither on proliferation, nor under chemotherapy treatments supporting these factors being fundamental in the control of HSCs. Summary/Conclusion: We characterized the secretome profile within 3D AML niche identifying PAI-1, CHI3L1, and PTX3 being upregulated. These factors were shown to modulate HSCs differentiation and proliferation capabilities in 3D providing a potential route for novel secretome-targeted strategies to favor the healthy hematopoiesis, and disadvantage AML progression.